b49bf2efdd846470f6c919b1a2918566 pmc_34209483.pdf 8064be62fc41be8159364e5c5f0de6bfed9342ae pmc_34209483.pdf b7b54958607a9782b854e240300d4eeb866a1d92888f91686fed64d60b20323a pmc_34209483.pdf Title: Antioxidant and Neuroprotective Properties of Non-Centrifugal Cane Sugar and Other Sugarcane Derivatives in an In Vitro Induced Parkinson’s Model Subject: Non-centrifugal cane sugar (NCS) is a traditional sweetener in most sugarcane regions of the world. In Colombia, this product has a socio-economic importance due to the extensive cultivation area and the high consumption rate per capita. NCS traditional processing involves consecutive stages of thermal processing that begin with juice extraction, clarification, evaporation, and finish with syrup crystallization into a solid commercial product, identified as NCS. Sugarcane is known to have a natural content of polyphenols, amino acids, vitamins, minerals, and complex sugars, some of which are reported as antioxidant and antiproliferative agents thought to be responsible for the product’s bioactive profile. There is evidence to suggest that traditional thermal processing to obtain NCS leads to a considerable decrease in the contents of these bioactive compounds, mainly due to uncontrolled process variables such as temperature. Accordingly, the aim of this study was to assess and compare the bioactivity of sugarcane (SC) derivatives produced under controlled thermal conditions versus the traditional method. To achieve this goal, we evaluated the cytotoxic, antioxidant, and neuroprotective effects of varying concentrations of SC derivatives in an in vitro induced Parkinson’s model. Results demonstrate non-cytotoxic activity on the cellular model by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and LDH assays, even at the highest tested concentration of 8 mg/mL, for all SC derivatives. The effect of SC derivatives on the induced oxidative stress model showed a biological reversion and recovering effect of the mitochondrial membrane potential and a halting of the progress into the early apoptosis phase. In conclusion, we demonstrated that the bioactive compounds present in SC derivatives obtained by a process under controlled temperature conditions are largely preserved, and even their biological activities are enhanced compared with SC derivatives obtained by the traditional thermal evaporation of SC-juice. Keywords: sugarcane; antioxidant; Parkinson’s disease; neuroprotective; mitochondrial membrane potential; non-centrifugal cane sugar Author: Javier Cifuentes, Vivian A. Salazar, Mónica Cuellar, María Claudia Castellanos, Jader Rodríguez, Juan C. Cruz and Carolina Muñoz-Camargo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jun 29 09:19:18 2021 CEST ModDate: Tue Jun 29 09:26:40 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 8711807 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YWWVQQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 15 0 KVKTZM+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 DENAWS+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 ZNFUSB+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 58 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 62 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 66 0 GZJNBV+CMSY10 Type 1 Builtin yes yes no 78 0 WABLSD+SFRM1000 Type 1 Custom yes yes no 82 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 96 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 99 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 102 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 109 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 113 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 135 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 138 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 144 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 147 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 157 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 160 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 166 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 169 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 180 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 183 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 189 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 192 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 202 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 205 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 211 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 214 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 224 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 227 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 233 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 236 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 246 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 249 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 255 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 258 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 269 0 NMDIFG+PalatinoLinotype CID TrueType Identity-H yes yes yes 272 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 278 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 281 0 NMDIFF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 292 0 NMDIFH+PalatinoLinotype TrueType WinAnsi yes yes no 295 0 NMDIFJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 298 0 RDUGED+VnURWPalladioL-Italic Type 1 Custom yes yes no 312 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 02:43:12 CEST RepresentationInformation: pmc_34209483.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:14:24 CEST Size: 8711807 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 584 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Sugarcane-Derivatives from Controlled Laboratory Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: SC Derivatives Sample Preparation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Quantification of Phenolic Content via the Folin Ciocalteu Method Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Identification and Quantification of Phenolic Compounds by Analytical HPLC-MS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Preparation of Phenolic Extracts Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Analytical Liquid Chromatography-Mass Spectroscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: SC Derivatives Free Radical Scavenging Antioxidant Activity Assessment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: DPPH Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: ABTS Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Cell Culture and Cytotoxicity of SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: MTT Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: LDH Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Neuroprotective Effects in SH-SY5Y Cell Parkinson’s Disease Model (PD-Induced Model) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: PD Model Cell Viability after Exposure to SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Apoptosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Apoptosis by Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Membrane Potential by Fluorescence Quantification and Confocal Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Membrane Potential by Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: MAO-B Activity Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Children: Item: Title: Polyphenolic Content and Free Radical Scavenging Properties SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Identification and Quantification of Phenolic Compounds by Analytical HPLC-MS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Cytotoxicity of SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Recovery Effect of a Rotenone PD-Induced Model Exposed to SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Mitochondrial Membrane Potential Recovery on the PD-Induced Model Treated withSC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Enzymatic Activity of Monoaminoxidase-B (MAO-B) in the PD-Induced Model Exposed to SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Apoptosis and Cell Cycle Arrest in the PD-Induced Model Exposed to SC Derivatives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Info: Title: Antioxidant and Neuroprotective Properties of Non-Centrifugal Cane Sugar and Other Sugarcane Derivatives in an In Vitro Induced Parkinson’s Model Author: Javier Cifuentes, Vivian A. Salazar, Mónica Cuellar, María Claudia Castellanos, Jader Rodríguez, Juan C. Cruz and Carolina Muñoz-Camargo Subject: Non-centrifugal cane sugar (NCS) is a traditional sweetener in most sugarcane regions of the world. In Colombia, this product has a socio-economic importance due to the extensive cultivation area and the high consumption rate per capita. NCS traditional processing involves consecutive stages of thermal processing that begin with juice extraction, clarification, evaporation, and finish with syrup crystallization into a solid commercial product, identified as NCS. Sugarcane is known to have a natural content of polyphenols, amino acids, vitamins, minerals, and complex sugars, some of which are reported as antioxidant and antiproliferative agents thought to be responsible for the product’s bioactive profile. There is evidence to suggest that traditional thermal processing to obtain NCS leads to a considerable decrease in the contents of these bioactive compounds, mainly due to uncontrolled process variables such as temperature. Accordingly, the aim of this study was to assess and compare the bioactivity of sugarcane (SC) derivatives produced under controlled thermal conditions versus the traditional method. To achieve this goal, we evaluated the cytotoxic, antioxidant, and neuroprotective effects of varying concentrations of SC derivatives in an in vitro induced Parkinson’s model. Results demonstrate non-cytotoxic activity on the cellular model by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and LDH assays, even at the highest tested concentration of 8 mg/mL, for all SC derivatives. The effect of SC derivatives on the induced oxidative stress model showed a biological reversion and recovering effect of the mitochondrial membrane potential and a halting of the progress into the early apoptosis phase. In conclusion, we demonstrated that the bioactive compounds present in SC derivatives obtained by a process under controlled temperature conditions are largely preserved, and even their biological activities are enhanced compared with SC derivatives obtained by the traditional thermal evaporation of SC-juice. Keywords: sugarcane; antioxidant; Parkinson’s disease; neuroprotective; mitochondrial membrane potential; non-centrifugal cane sugar Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jun 29 09:19:18 CEST 2021 ModDate: Tue Jun 29 09:26:40 CEST 2021 ID: 0xa287c6376d5d3986d452b0e4a01dda14, 0xa287c6376d5d3986d452b0e4a01dda14 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: WABLSD+SFRM1000 FontSubset: true FirstChar: 180 LastChar: 180 FontDescriptor: FontName: WABLSD+SFRM1000 Flags: Symbolic FontBBox: -189, -321, 1456, 937 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: KVKTZM+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: KVKTZM+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DENAWS+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: DENAWS+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: RDUGED+VnURWPalladioL-Italic FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: RDUGED+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YWWVQQ+VnURWPalladioL-Bold FontSubset: true FirstChar: 189 LastChar: 226 FontDescriptor: FontName: YWWVQQ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZNFUSB+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: ZNFUSB+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: GZJNBV+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: GZJNBV+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: MWHIBA+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: MWHIBA+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 175, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 121, 643, 132, 656 Annotation: Subtype: Link Rect: 235, 643, 246, 656 Annotation: Subtype: Link Rect: 326, 643, 337, 656 Annotation: Subtype: Link Rect: 106, 630, 117, 644 Annotation: Subtype: Link Rect: 269, 630, 280, 644 Annotation: Subtype: Link Rect: 121, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 334, 137, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 131, 17, 277, 28 Annotation: Subtype: Link Rect: 390, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 458, 87, 477 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 208, 661, 215, 670 ActionDest: 309 Annotation: Subtype: Link Rect: 215, 661, 222, 670 ActionDest: 309 Annotation: Subtype: Link Rect: 285, 624, 292, 633 ActionDest: 309 Annotation: Subtype: Link Rect: 414, 561, 421, 570 ActionDest: 309 Annotation: Subtype: Link Rect: 424, 561, 431, 570 ActionDest: 309 Annotation: Subtype: Link Rect: 480, 536, 487, 545 ActionDest: 309 Annotation: Subtype: Link Rect: 380, 511, 387, 520 ActionDest: 309 Annotation: Subtype: Link Rect: 390, 511, 402, 520 ActionDest: 309 Annotation: Subtype: Link Rect: 192, 486, 199, 495 ActionDest: 309 Annotation: Subtype: Link Rect: 325, 473, 332, 482 ActionDest: 309 Annotation: Subtype: Link Rect: 332, 473, 339, 482 ActionDest: 309 Annotation: Subtype: Link Rect: 340, 473, 352, 482 ActionDest: 309 Annotation: Subtype: Link Rect: 524, 448, 531, 457 ActionDest: 309 Annotation: Subtype: Link Rect: 531, 448, 543, 457 ActionDest: 309 Annotation: Subtype: Link Rect: 544, 448, 556, 457 ActionDest: 309 Annotation: Subtype: Link Rect: 544, 372, 556, 382 ActionDest: 316 Annotation: Subtype: Link Rect: 311, 310, 323, 319 ActionDest: 316 Annotation: Subtype: Link Rect: 324, 310, 336, 319 ActionDest: 316 Annotation: Subtype: Link Rect: 442, 260, 454, 269 ActionDest: 316 Annotation: Subtype: Link Rect: 506, 109, 518, 118 ActionDest: 316 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 353, 647, 365, 656 ActionDest: 316 Annotation: Subtype: Link Rect: 393, 584, 405, 593 ActionDest: 316 Annotation: Subtype: Link Rect: 405, 584, 417, 593 ActionDest: 316 Annotation: Subtype: Link Rect: 489, 572, 501, 581 ActionDest: 316 Annotation: Subtype: Link Rect: 235, 522, 247, 530 ActionDest: 316 Annotation: Subtype: Link Rect: 247, 521, 259, 530 ActionDest: 316 Annotation: Subtype: Link Rect: 205, 79, 212, 90 ActionDest: 90 Page: Sequence: 4 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 248, 672, 260, 681 ActionDest: 316 Annotation: Subtype: Link Rect: 289, 530, 296, 542 ActionDest: 90 Annotation: Subtype: Link Rect: 206, 101, 213, 114 ActionDest: 90 Annotation: Subtype: Link Rect: 220, 104, 232, 113 ActionDest: 309 Annotation: Subtype: Link Rect: 232, 104, 244, 113 ActionDest: 316 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 422, 560, 429, 572 ActionDest: 90 Annotation: Subtype: Link Rect: 187, 525, 199, 534 ActionDest: 316 Annotation: Subtype: Link Rect: 303, 323, 310, 337 ActionDest: 90 Annotation: Subtype: Link Rect: 549, 137, 556, 149 ActionDest: 90 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 207, 604, 214, 616 ActionDest: 90 Annotation: Subtype: Link Rect: 477, 581, 489, 590 ActionDest: 316 Annotation: Subtype: Link Rect: 490, 581, 502, 590 ActionDest: 316 Annotation: Subtype: Link Rect: 424, 544, 436, 553 ActionDest: 316 Annotation: Subtype: Link Rect: 438, 352, 445, 364 ActionDest: 90 Annotation: Subtype: Link Rect: 475, 305, 487, 314 ActionDest: 316 Annotation: Subtype: Link Rect: 530, 251, 537, 263 ActionDest: 90 Annotation: Subtype: Link Rect: 544, 254, 556, 263 ActionDest: 316 Annotation: Subtype: Link Rect: 494, 100, 501, 112 ActionDest: 90 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 430, 556, 437, 568 ActionDest: 90 Annotation: Subtype: Link Rect: 444, 559, 456, 568 ActionDest: 316 Annotation: Subtype: Link Rect: 227, 493, 234, 505 ActionDest: 90 Annotation: Subtype: Link Rect: 191, 180, 198, 190 ActionDest: 126 Annotation: Subtype: Link Rect: 287, 178, 294, 190 ActionDest: 126 Annotation: Subtype: Link Rect: 536, 118, 543, 127 ActionDest: 309 Annotation: Subtype: Link Rect: 544, 118, 556, 127 ActionDest: 316 Annotation: Subtype: Link Rect: 390, 90, 396, 102 ActionDest: 126 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 304, 722, 316, 731 ActionDest: 316 Annotation: Subtype: Link Rect: 476, 533, 483, 545 ActionDest: 126 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 516, 644, 523, 656 ActionDest: 129 Annotation: Subtype: Link Rect: 540, 178, 547, 190 ActionDest: 151 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 447, 337, 459, 346 ActionDest: 316 Annotation: Subtype: Link Rect: 497, 334, 504, 346 ActionDest: 174 Annotation: Subtype: Link Rect: 388, 236, 400, 245 ActionDest: 316 Annotation: Subtype: Link Rect: 439, 234, 446, 246 ActionDest: 174 Page: Sequence: 12 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 269, 602, 281, 611 ActionDest: 316 Annotation: Subtype: Link Rect: 282, 602, 294, 611 ActionDest: 316 Annotation: Subtype: Link Rect: 508, 590, 520, 599 ActionDest: 316 Annotation: Subtype: Link Rect: 520, 590, 532, 599 ActionDest: 316 Annotation: Subtype: Link Rect: 549, 562, 556, 574 ActionDest: 218 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 428, 386, 440, 395 ActionDest: 316 Annotation: Subtype: Link Rect: 440, 386, 452, 395 ActionDest: 316 Annotation: Subtype: Link Rect: 404, 358, 411, 370 ActionDest: 240 Annotation: Subtype: Link Rect: 274, 235, 286, 244 ActionDest: 316 Annotation: Subtype: Link Rect: 286, 235, 298, 244 ActionDest: 316 Annotation: Subtype: Link Rect: 233, 222, 245, 231 ActionDest: 316 Annotation: Subtype: Link Rect: 245, 222, 257, 231 ActionDest: 316 Annotation: Subtype: Link Rect: 404, 210, 416, 219 ActionDest: 316 Annotation: Subtype: Link Rect: 453, 207, 460, 221 ActionDest: 263 Annotation: Subtype: Link Rect: 464, 184, 476, 194 ActionDest: 316 Annotation: Subtype: Link Rect: 476, 184, 488, 194 ActionDest: 316 Annotation: Subtype: Link Rect: 491, 184, 503, 194 ActionDest: 316 Annotation: Subtype: Link Rect: 355, 159, 366, 168 ActionDest: 316 Annotation: Subtype: Link Rect: 367, 160, 379, 168 ActionDest: 316 Annotation: Subtype: Link Rect: 189, 122, 201, 131 ActionDest: 316 Annotation: Subtype: Link Rect: 202, 122, 214, 131 ActionDest: 316 Annotation: Subtype: Link Rect: 313, 107, 320, 119 ActionDest: 263 Annotation: Subtype: Link Rect: 424, 107, 431, 119 ActionDest: 263 Annotation: Subtype: Link Rect: 200, 56, 207, 68 ActionDest: 286 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 306, 735, 318, 744 ActionDest: 316 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 259, 284, 265, 295 ActionDest: 174 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 196, 73, 203, 82 ActionDest: 309 Annotation: Subtype: Link Rect: 203, 73, 215, 82 ActionDest: 309 Annotation: Subtype: Link Rect: 216, 73, 228, 82 ActionDest: 316 Annotation: Subtype: Link Rect: 228, 73, 240, 82 ActionDest: 320 Annotation: Subtype: Link Rect: 243, 73, 255, 82 ActionDest: 320 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 289, 722, 301, 731 ActionDest: 309 Annotation: Subtype: Link Rect: 301, 722, 313, 731 ActionDest: 320 Annotation: Subtype: Link Rect: 313, 722, 325, 731 ActionDest: 320 Annotation: Subtype: Link Rect: 195, 670, 202, 681 ActionDest: 126 Annotation: Subtype: Link Rect: 209, 672, 221, 681 ActionDest: 320 Annotation: Subtype: Link Rect: 224, 672, 236, 681 ActionDest: 320 Annotation: Subtype: Link Rect: 544, 659, 556, 669 ActionDest: 320 Annotation: Subtype: Link Rect: 294, 572, 306, 581 ActionDest: 320 Annotation: Subtype: Link Rect: 307, 572, 319, 581 ActionDest: 320 Annotation: Subtype: Link Rect: 404, 506, 411, 518 ActionDest: 129 Annotation: Subtype: Link Rect: 397, 481, 404, 493 ActionDest: 151 Annotation: Subtype: Link Rect: 442, 481, 449, 493 ActionDest: 151 Annotation: Subtype: Link Rect: 544, 433, 556, 443 ActionDest: 320 Annotation: Subtype: Link Rect: 549, 393, 556, 405 ActionDest: 174 Annotation: Subtype: Link Rect: 420, 371, 432, 380 ActionDest: 316 Annotation: Subtype: Link Rect: 433, 371, 445, 380 ActionDest: 320 Annotation: Subtype: Link Rect: 200, 318, 207, 330 ActionDest: 218 Annotation: Subtype: Link Rect: 479, 308, 491, 317 ActionDest: 316 Annotation: Subtype: Link Rect: 544, 295, 556, 304 ActionDest: 316 Annotation: Subtype: Link Rect: 544, 270, 556, 279 ActionDest: 316 Annotation: Subtype: Link Rect: 215, 245, 227, 254 ActionDest: 316 Annotation: Subtype: Link Rect: 381, 230, 388, 242 ActionDest: 286 Annotation: Subtype: Link Rect: 549, 180, 556, 192 ActionDest: 263 Annotation: Subtype: Link Rect: 201, 142, 208, 154 ActionDest: 286 Annotation: Subtype: Link Rect: 422, 82, 429, 91 ActionDest: 309 Annotation: Subtype: Link Rect: 430, 82, 442, 91 ActionDest: 320 Annotation: Subtype: Link Rect: 442, 82, 454, 91 ActionDest: 320 Annotation: Subtype: Link Rect: 455, 82, 467, 91 ActionDest: 320 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 186, 722, 198, 731 ActionDest: 316 Annotation: Subtype: Link Rect: 199, 722, 211, 731 ActionDest: 320 Annotation: Subtype: Link Rect: 211, 722, 223, 731 ActionDest: 320 Annotation: Subtype: Link Rect: 544, 634, 556, 643 ActionDest: 320 Annotation: Subtype: Link Rect: 248, 622, 260, 631 ActionDest: 320 Annotation: Subtype: Link Rect: 387, 572, 399, 581 ActionDest: 320 Annotation: Subtype: Link Rect: 399, 572, 411, 581 ActionDest: 320 Annotation: Subtype: Link Rect: 412, 572, 424, 581 ActionDest: 320 Annotation: Subtype: Link Rect: 384, 408, 396, 417 ActionDest: 309 Annotation: Subtype: Link Rect: 397, 408, 409, 417 ActionDest: 309 Annotation: Subtype: Link Rect: 409, 408, 421, 417 ActionDest: 320 Annotation: Subtype: Link Rect: 206, 346, 218, 355 ActionDest: 320 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 166, 336, 445, 347 Annotation: Subtype: Link Rect: 441, 324, 479, 335 Annotation: Subtype: Link Rect: 176, 301, 320, 312 Annotation: Subtype: Link Rect: 95, 279, 133, 289 Annotation: Subtype: Link Rect: 257, 221, 294, 232 Annotation: Subtype: Link Rect: 256, 187, 294, 197 Annotation: Subtype: Link Rect: 506, 163, 543, 174 Annotation: Subtype: Link Rect: 118, 141, 155, 151 Annotation: Subtype: Link Rect: 162, 141, 198, 151 Annotation: Subtype: Link Rect: 394, 117, 432, 128 Annotation: Subtype: Link Rect: 445, 94, 483, 105 Annotation: Subtype: Link Rect: 473, 71, 510, 82 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 476, 721, 513, 732 Annotation: Subtype: Link Rect: 56, 686, 286, 697 Annotation: Subtype: Link Rect: 455, 663, 492, 674 Annotation: Subtype: Link Rect: 516, 652, 554, 663 Annotation: Subtype: Link Rect: 164, 629, 201, 640 Annotation: Subtype: Link Rect: 303, 606, 341, 617 Annotation: Subtype: Link Rect: 347, 606, 383, 617 Annotation: Subtype: Link Rect: 188, 584, 226, 594 Annotation: Subtype: Link Rect: 232, 584, 268, 594 Annotation: Subtype: Link Rect: 506, 560, 544, 571 Annotation: Subtype: Link Rect: 59, 526, 97, 536 Annotation: Subtype: Link Rect: 350, 502, 388, 513 Annotation: Subtype: Link Rect: 168, 480, 205, 490 Annotation: Subtype: Link Rect: 445, 468, 483, 479 Annotation: Subtype: Link Rect: 95, 434, 133, 444 Annotation: Subtype: Link Rect: 246, 410, 283, 421 Annotation: Subtype: Link Rect: 200, 388, 238, 398 Annotation: Subtype: Link Rect: 274, 365, 312, 375 Annotation: Subtype: Link Rect: 519, 341, 557, 352 Annotation: Subtype: Link Rect: 59, 331, 95, 341 Annotation: Subtype: Link Rect: 453, 307, 490, 318 Annotation: Subtype: Link Rect: 497, 307, 533, 318 Annotation: Subtype: Link Rect: 404, 284, 442, 295 Annotation: Subtype: Link Rect: 458, 261, 496, 272 Annotation: Subtype: Link Rect: 519, 238, 557, 249 Annotation: Subtype: Link Rect: 59, 227, 95, 237 Annotation: Subtype: Link Rect: 314, 203, 351, 214 Annotation: Subtype: Link Rect: 127, 181, 164, 191 Annotation: Subtype: Link Rect: 131, 158, 169, 168 Annotation: Subtype: Link Rect: 233, 123, 271, 134 Annotation: Subtype: Link Rect: 354, 100, 391, 111 Annotation: Subtype: Link Rect: 398, 100, 434, 111 Annotation: Subtype: Link Rect: 519, 77, 557, 88 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 334, 721, 371, 732 Annotation: Subtype: Link Rect: 95, 687, 133, 697 Annotation: Subtype: Link Rect: 389, 663, 427, 674 Annotation: Subtype: Link Rect: 73, 630, 110, 640 Annotation: Subtype: Link Rect: 104, 607, 142, 617 Annotation: Subtype: Link Rect: 148, 607, 184, 617 Annotation: Subtype: Link Rect: 366, 583, 404, 594 Annotation: Subtype: Link Rect: 394, 560, 431, 571 Annotation: Subtype: Link Rect: 438, 560, 474, 571 Annotation: Subtype: Link Rect: 247, 537, 285, 548 Annotation: Subtype: Link Rect: 291, 537, 327, 548 Annotation: Subtype: Link Rect: 90, 503, 128, 513 Annotation: Subtype: Link Rect: 134, 503, 170, 513 Annotation: Subtype: Link Rect: 187, 479, 560, 490 Annotation: Subtype: Link Rect: 56, 468, 245, 479 Annotation: Subtype: Link Rect: 355, 445, 392, 456 Annotation: Subtype: Link Rect: 190, 422, 228, 433 Annotation: Subtype: Link Rect: 234, 422, 270, 433 Annotation: Subtype: Link Rect: 520, 399, 557, 410 Annotation: Subtype: Link Rect: 59, 377, 97, 387 Annotation: Subtype: Link Rect: 103, 377, 139, 387 Annotation: Subtype: Link Rect: 427, 365, 465, 375 Annotation: Subtype: Link Rect: 300, 341, 338, 352 Annotation: Subtype: Link Rect: 519, 307, 557, 318 Annotation: Subtype: Link Rect: 59, 273, 97, 283 Annotation: Subtype: Link Rect: 103, 273, 139, 283 Annotation: Subtype: Link Rect: 140, 238, 178, 249 Annotation: Subtype: Link Rect: 184, 238, 220, 249 Annotation: Subtype: Link Rect: 422, 215, 459, 226 Checksum: d5cd42ea Type: CRC32 Checksum: b49bf2efdd846470f6c919b1a2918566 Type: MD5 Checksum: 8064be62fc41be8159364e5c5f0de6bfed9342ae Type: SHA-1